a.**Methylation of DNA**

It is the renowned epigenetic modification that is well studied in cancer, and lot of interest has been generated in DNA methylation in the framework of diabetes and its related complications. In detail, DNA undergoes methylation at 5th position of CpG dinucleotides and form 5-methylcytosine, which is a post-replicative mechanism. DNA methylation is extremely dynamic process in the progress of a disease, which tends to alter related gene expressions. These alterations can be reversed by external stimuli. Commonly repression of a gene takes place due to addition of methyl groups at promoter region on DNA, while methylation at gene bodies may regulate their transcription during elongation and also during alternative splicing [31].

DNA methyl transferases (DNMTs) are known to catalyze DNA methylation reaction which, in freshly synthesized DNA, methylates CpG dinucleotides. Hence, to sustain DNA methylation in proliferating cells, DNMTs are vital. Throughout the embryonic development, for *de novo* methylation, presence of DNMT-3a and -3b enzymes is obligatory [47]. The molecular effects of DNA methylation were interceded by a group of methyl binding domain (MBD) proteins. Out of these, merely MBD2 alone is identifiable for methyl-CpG positions, which guides the interaction of methylated DNA to a multifaceted complex encompassing nucleosome remodeling and histone deacetylases (HDACs) bustles, thereby conducting silencing of a gene [48]. DNA methylation is commonly studied by various methods including methylation specific PCR (MS-PCR), methylation sensitive high resolution melt

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*Diabetes Microvascular Complications: An Overview of Epigenetic Modifications*

of methylation is linked with time towards the progression of DN [50].

between MD and cardiovascular disorders [56].

It has been demonstrated that the promoter of human ACE gene, the most important and widely studied gene in pathophysiology of DN, harbor CpG islands. ACE transcription and expression levels were also observed to be influenced by methylation in its promoter region both in vivo and in vitro [54]. The magnitude of epigenetic alterations, particularly DNA methylation, has been shown to correlate with ACE activity levels [54, 55]. These studies demonstrated an increase in ACE activity with hypomethylation of ACE gene promoter. Also, a relation between epigenetics of ACE gene and I/D polymorphism has been suggested, where decreased DNA methylation in 3 CpG sites of ACE gene was observed in low birth weight (LBW) children with DD genotype although this has not been reported directly in DN patients [55]. Global DNA methylation variations were also observed to be associated with albuminuria in a recent study [52]. Additionally alterations in DNA methylation of ACE promoter are suggested to be a fundamental cause of major depression (MD) and a shared pathogenic factor for bi-directional connection

Apart from the importance of DNA methylation in DN, their role in DR is not clear, however, DNA methylation has been shown to control the expressions of many genes associated with retinal homeostasis. Previous studies have shown the link of DR development and DNA methylation, which indicates that DR may be associated with epigenetic alterations. In this connection, a GWAS between PDR and healthy controls was conducted in PBMC'S sample and out of 349 identified methylated sites, only 17 genes were observed to be hypermethylated [57]. They assumed that PBMCs could be used as a predictor for diabetic retinopathy. Another study evaluated global DNA methylation levels in blood leukocytes in persons with and without retinopathy [58]. They found a significantly higher global methylation levels in patients with DR than those without DR. These changes were seen to be

curve (MS-HRM), immunoprecipitation or sequencing approaches. Advantage of MS-HRM is that it offers a low-cost and rapid method for the detection of even low

Earlier exposure of target cells to high glucose can result in a 'metabolic memory' which results in persistence of its detrimental effects long after glucose stabilization. Diabetes-induced altered epigenetic mechanisms, resulting in modified gene expression in target cells can lead to diabetes-associated complications, such as diabetic nephropathy [39]. In the pathogenesis of DN and ESRD, DNAme (DNA methylation) has been explored by several studies via studying differentially methylated genes related to DN [31, 49, 50]. In a genome-wide methylation analysis (GWAS), significant alterations in DNA methylation in DN patients as compared to control were reported at 19 CpG sites that were found to be associated with the risk of DN. They also correlated the degree of methylation with time to development of DN [50]. In DNA isolated from the saliva of type 2 diabetic patients with end-stage kidney disease (ESRD), differentially site-specific methylation of DNA was recorded at 187 gene targets in comparison to those without ESRD [49]. DN patients have altered DNA methylation at important key gene promoters in comparison to those without DN [50]. However, studies in DN animal models or in renal cells under hyperglycemic conditions were not competent to show any significant changes in DNA methylation patterns [51]. In patients having type 2 diabetes with diabetic nephropathy, global DNA methylation variations were also observed to be associated with albuminuria in a recent study [52]. Noteworthy alterations in histone and DNA methylation patterns were observed to be present in peripheral blood mononuclear cells (PBMCs) of patients with membranous nephropathy [53]. Genome-wide DNA methylation study also depicted modifications in differential DNA methylation profiles among type 1 diabetes patients with or without nephropathy, where degree

*DOI: http://dx.doi.org/10.5772/intechopen.94642*

levels of methylation at gene promoters.

#### *Diabetes Microvascular Complications: An Overview of Epigenetic Modifications DOI: http://dx.doi.org/10.5772/intechopen.94642*

curve (MS-HRM), immunoprecipitation or sequencing approaches. Advantage of MS-HRM is that it offers a low-cost and rapid method for the detection of even low levels of methylation at gene promoters.

Earlier exposure of target cells to high glucose can result in a 'metabolic memory' which results in persistence of its detrimental effects long after glucose stabilization. Diabetes-induced altered epigenetic mechanisms, resulting in modified gene expression in target cells can lead to diabetes-associated complications, such as diabetic nephropathy [39]. In the pathogenesis of DN and ESRD, DNAme (DNA methylation) has been explored by several studies via studying differentially methylated genes related to DN [31, 49, 50]. In a genome-wide methylation analysis (GWAS), significant alterations in DNA methylation in DN patients as compared to control were reported at 19 CpG sites that were found to be associated with the risk of DN. They also correlated the degree of methylation with time to development of DN [50]. In DNA isolated from the saliva of type 2 diabetic patients with end-stage kidney disease (ESRD), differentially site-specific methylation of DNA was recorded at 187 gene targets in comparison to those without ESRD [49]. DN patients have altered DNA methylation at important key gene promoters in comparison to those without DN [50]. However, studies in DN animal models or in renal cells under hyperglycemic conditions were not competent to show any significant changes in DNA methylation patterns [51]. In patients having type 2 diabetes with diabetic nephropathy, global DNA methylation variations were also observed to be associated with albuminuria in a recent study [52]. Noteworthy alterations in histone and DNA methylation patterns were observed to be present in peripheral blood mononuclear cells (PBMCs) of patients with membranous nephropathy [53]. Genome-wide DNA methylation study also depicted modifications in differential DNA methylation profiles among type 1 diabetes patients with or without nephropathy, where degree of methylation is linked with time towards the progression of DN [50].

It has been demonstrated that the promoter of human ACE gene, the most important and widely studied gene in pathophysiology of DN, harbor CpG islands. ACE transcription and expression levels were also observed to be influenced by methylation in its promoter region both in vivo and in vitro [54]. The magnitude of epigenetic alterations, particularly DNA methylation, has been shown to correlate with ACE activity levels [54, 55]. These studies demonstrated an increase in ACE activity with hypomethylation of ACE gene promoter. Also, a relation between epigenetics of ACE gene and I/D polymorphism has been suggested, where decreased DNA methylation in 3 CpG sites of ACE gene was observed in low birth weight (LBW) children with DD genotype although this has not been reported directly in DN patients [55]. Global DNA methylation variations were also observed to be associated with albuminuria in a recent study [52]. Additionally alterations in DNA methylation of ACE promoter are suggested to be a fundamental cause of major depression (MD) and a shared pathogenic factor for bi-directional connection between MD and cardiovascular disorders [56].

Apart from the importance of DNA methylation in DN, their role in DR is not clear, however, DNA methylation has been shown to control the expressions of many genes associated with retinal homeostasis. Previous studies have shown the link of DR development and DNA methylation, which indicates that DR may be associated with epigenetic alterations. In this connection, a GWAS between PDR and healthy controls was conducted in PBMC'S sample and out of 349 identified methylated sites, only 17 genes were observed to be hypermethylated [57]. They assumed that PBMCs could be used as a predictor for diabetic retinopathy. Another study evaluated global DNA methylation levels in blood leukocytes in persons with and without retinopathy [58]. They found a significantly higher global methylation levels in patients with DR than those without DR. These changes were seen to be

*Type 2 Diabetes - From Pathophysiology to Cyber Systems*

*non-coding RNAs, miRNAs, micro RNAs.*

*Framework of inheritable epigenetic modifications.*

a.**Methylation of DNA**

**Figure 3.**

tive splicing [31].

*stimuli via regulation of miRNAs, thus directing several pathophysiological outcomes. DNMTs, DNA methyl transferases; HATs, Histone acetyl transferases; HDACs, Histone deacetylases; HMTs, Histone methyl transferases; HDMs, Histone demethylases; ncRNAs,* 

It is the renowned epigenetic modification that is well studied in cancer, and lot of interest has been generated in DNA methylation in the framework of diabetes and its related complications. In detail, DNA undergoes methylation at 5th position of CpG dinucleotides and form 5-methylcytosine, which is a post-replicative mechanism. DNA methylation is extremely dynamic process in the progress of a disease, which tends to alter related gene expressions. These alterations can be reversed by external stimuli. Commonly repression of a gene takes place due to addition of methyl groups at promoter region on DNA, while methylation at gene bodies may regulate their transcription during elongation and also during alterna-

DNA methyl transferases (DNMTs) are known to catalyze DNA methylation reaction which, in freshly synthesized DNA, methylates CpG dinucleotides. Hence, to sustain DNA methylation in proliferating cells, DNMTs are vital. Throughout the embryonic development, for *de novo* methylation, presence of DNMT-3a and -3b enzymes is obligatory [47]. The molecular effects of DNA methylation were interceded by a group of methyl binding domain (MBD) proteins. Out of these, merely MBD2 alone is identifiable for methyl-CpG positions, which guides the interaction of methylated DNA to a multifaceted complex encompassing nucleosome remodeling and histone deacetylases (HDACs) bustles, thereby conducting silencing of a gene [48]. DNA methylation is commonly studied by various methods including methylation specific PCR (MS-PCR), methylation sensitive high resolution melt

**178**

progressive from non-DR stage to NPDR and eventually to PDR and were independent of hyperglycemia, dyslipidemia, diabetes duration and person's blood pressure. Binding of polymerase gamma 1 (POLG1) to mtDNA (mitochondrial DNA) also results in compromised transcriptional activity as a result of hypermethylation at promoter region of DNA polymerase gamma 1 (POLG1) in the hyperglycemic environment [42]. This study was conducted in rat model of diabetes which showed that the mitochondrial damage in retina of diabetic rats could be diminished/controlled by maintaining stable glycemic control for longer time periods or therapy that targets directly DNA methylation. However, it does not benefit DNA methylation machinery by the reversal of hyper-glycemic environment for shorter duration [59]. In people with diabetes mellitus, it has been seen that activity of Dnmt1 enzyme was elevated in retinal and its capillary cells. However, this was not observed with Dnmt-3a or Dnmt-3b [60, 61]. Similar differential DNA methylation patterns were also observed in persons with PDR [57].
